Biology subjects

roche, s.

Publications and source records attributed to roche, s..

6 recordsLinked to original sources

bioRxiv2026

Progastrin activates colonic fibroblasts and induces a paracrine pro-migratory program in colorectal cancer cells

PurposeProgastrin, aberrantly expressed in colorectal cancer (CRC), is an established trophic factor for tumour epithelial cells. Whether it also promotes CRC progression by reprogramming stromal fibroblasts remains unclear. We investigated progastrin-induced colonic fibroblast activation and its functional consequences on CRC cell migration. MethodsFibroblast activation was assessed in the colonic mucosa of hGAS mice and in the human normal colonic fibroblast line CCD18Co exposed to synthetic progastrin. The impact of tumour-derived progastrin on epithelial cell motility was analysed using HCT116 cells expressing a control shRNA (shLuc) or a progastrin-targeting shRNA (shPG) in transwell migration assays performed with or without fibroblasts. Candidate paracrine mediators were evaluated by RT-qPCR, ELISA and neutralization experiments, and signalling was interrogated using the PI3K inhibitor LY294002. ResultsColonic fibroblasts from hGAS mice displayed stromal FAP and SMA expression, indicating fibroblast activation in vivo. In CCD18Co cells, progastrin increased FAP and SMA protein levels. Fibroblasts enhanced HCT116 cell migration. This effect was stronger when tumour cells expressed progastrin or when fibroblasts were preconditioned by progastrin-producing HCT116 cells. Progastrin induced CXCL12/SDF-1 and CXCL8/IL-8 expression and secretion by fibroblasts, and neutralization of either chemokine abrogated the additional migratory effect conferred by progastrin-activated fibroblasts. Progastrin triggered sustained Akt phosphorylation in fibroblasts, while PI3K inhibition suppressed CXCL12 and CXCL8 secretion and abolished fibroblast-dependent tumour cell migration. ConclusionThese data identify a stromal dimension of progastrin signalling in CRC and support a model in which tumour-derived progastrin activates colonic fibroblasts and elicits a PI3K/Akt-dependent paracrine programme that enhances CRC cell migration.

cancer biology↗

bioRxiv2025

Structures of vesicular stomatitis virus glycoprotein G alone and in complex with a neutralizing antibody

VSV G mediates viral entry via endocytosis. In the endosome, G undergoes a pH-dependent conformational change from pre- to post-fusion state, catalyzing membrane fusion. So far, no complete structure of G has been reported. We report cryo-EM structures of G, isolated from virions using detergent, alone and in complex with neutralizing antibody FAb that binds G in all conformations. The post-fusion structure reveals novel details about the organization of the C-terminal part of the ectodomain, showing that it undergoes conformational rearrangement and stabilizes the post-fusion trimer by nesting into a groove between adjacent fusion domains. The fusion loops are visible inside the micelle, which is not the case of the transmembrane domains, suggesting that they are rather mobile. Structures of G-FAb complex show that the epitope belongs to a conserved antigenic site. This work has potential implications for vaccine development and oncolytic virotherapy.

biochemistry↗

bioRxiv2024

Lipid-driven SRC self-association modulates its transformation capacity

Src tyrosine kinase is anchored to the plasma membrane, however, the contribution of membrane lipids to its regulation remains elusive. Here we report Src self-association through a lysine cluster in the Src SH4 region mediated by lipids in human cells and in vitro. Mutation of the lysine cluster to arginine modulates Src self-association and its transforming function in human cells. This mechanism may also apply to other membrane-associated signaling proteins that have similar lysine clusters in their unstructured regions. Lipid-anchored micron-sized condensates of full length Src are formed in supported homogeneous lipid bilayers (i.e independently of lipid phase separation). Condensates are also formed by the purified Src N-terminal regulatory element, including the myristoylated SH4 domain, the intrinsically disordered Unique domain and the globular SH3 domain. The isolated SH4 domain forms small protein-lipid clusters, but not micron-size condensates. Our findings reveal lipid-mediated kinase self-association as an additional mechanism for Src regulation. Significance StatementCondensate formation, often associated to intrinsically disordered protein regions is emerging as a widespread regulatory mechanism. Protein phase separation has been linked to the regulation of kinases recruited to condensates formed by other proteins. Here we show i) that the N-terminal disordered region of Src kinase drives lipid-mediated self-association to form condensates on the cell surface, ii) a lysine cluster in the N-terminal SH4 domain is essential for this process, iii) mutation of these lysine residues to arginine increases self-association in vitro and in vivo, and iv) self-association of Src results in enhanced transforming capacity in human cells. Similar lysine clusters are present in juxtamembrane regions suggest a general mechanism for modulating protein-protein interactions by lipids. The article shows the formation of condensates by Src on supported lipid membranes mediated by lipids, dependent of a conserved lysine cluster in its SH4 domain, and modulating Srcs transforming capacity. O_LIFull length Src and truncated variants including its myristoylated SH4 domain self-associate on the surface of homogeneous lipid bilayers. C_LIO_LIThe interaction of a lysine cluster in SH4 domain with lipids modulates Src self-association C_LIO_LIAtomic force microscopy shows the formation of micron-size condensates and smaller clusters on the surface of supported lipid bilayers, depending on the lipid composition or the presence of mutations in the lysine cluster C_LIO_LISrc self-association is confirmed in human cells and the Src transforming function of Src in human cells is modulated by lipid-mediated self-association C_LI

molecular biology↗

bioRxiv2021

SHED-dependent oncogenic signalling of the PEAK3 pseudo-kinase

The PEAK1 and pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal by a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signalling mechanism remains unclear. Here, we found that although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, PEAK3 protein is strongly expressed in human haematopoietic cells, and is upregulated in acute myeloid leukaemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signalling, independent of growth factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signalling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2 to promote AKT signalling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signalling proteins to promote oncogenesis.

cancer biology↗

bioRxiv2021

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

The membrane anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Srcs oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

cancer biology↗

bioRxiv2017

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

biochemistry↗